不同的引导肽对丝状噬菌体基因8蛋白展示外源多肽的影响

为了研究不同的引导肽对在基因8蛋白上展示外源多肽的影响,利用基因工程方法将一短肽插入基因8蛋白的N端,并将引导多肽去除或用基因3蛋白的引导肽序列替代基因8的引导肽序列.采用放射性脉冲追踪技术检测不同的引导肽对在基因8蛋白上展示外源多肽的作用.结果表明,无引导肽序列的引导,蛋白前体无法向成熟蛋白转化.基因3蛋白的引导肽可以引导展示有外源多肽的基因8蛋白,完成从前体向成熟蛋白的转化,但转化率明显下降.研究结果对阐明噬菌体外壳蛋白在大肠杆菌中的跨膜机制具有重要的意义.     

功能多肽在肿瘤治疗中的应用及研究进展

多肽具有生物相容性好,功能多样化,生物体内响应性高及合成修饰方法简单易行等优点,已被广泛用于构建靶向药物传递系统。以具有靶向功能和刺激响应性的多肽为基础构建的药物传递系统,能够将药物定向地运送到肿瘤区域。药物传递系统到达肿瘤组织后,在肿瘤组织特殊微环境或外源刺激下,实现药物的精准释放。这种具有特异性肿瘤靶向和刺激响应型的多肽载体可以最大程度地提高药物的抗肿瘤效果,降低药物的毒副作用。本文简要介绍了常用的靶向多肽和刺激响应型多肽,并讨论了基于功能型多肽的药物载体在肿瘤治疗方面的应用。     

酶解与质谱结合分析芋螺毒素Bt ⅢA 的C-端序列

虽然建立在Edman降解化学法基础上的自动化多肽蛋白N-端测序技术已经日益成熟,但有时也会遇到难以克服的困难.比如,多肽蛋白的N-末端被封闭、随着测序循环的进行导致吸附在惰性载体上的多肽链流失而难以得到完整的多肽序列等,这些都需要借助C-端序列分析来解决.     

芜菁花叶病毒外壳蛋白基因的克隆及其植物表达载体的构建

从感病的萝卜叶片中得到芜菁花叶病毒(TuMV)的分离物,以oligo(dT)为引物,合成了其基因组RNA的互补DNA,并克隆到载体λ-ZAPⅡ中。通过单链cDNA探针杂交和DNA末端序列分析找出含有poly-A尾巴的阳性克隆。我们对一个含有1429个碱基插入片断的克隆进行了序列分析,根据芜菁花叶病毒外壳蛋白分子量的大小和马铃薯Y病毒组蛋白质加工位点氨基酸序列的保守性,确定了外壳蛋白基因的5′末端。利用PCR方法对其5′末端进行改造,加进起始密码ATG和两个限制性内切酶位点。通过基因重组操作,将该基因插入到双元载体pBI121的衍生物pBIN437中,得到芜菁花叶病毒外壳蛋白基因的植物表达载体。     

刺激响应型多肽的研究及其在肿瘤诊疗中的应用

多肽因其独特的理化性能,如生物相容性好,合成修饰方法简单易行,功能多样化和生物体内响应性高等优点,已被广泛用于构建刺激响应型肿瘤诊疗体系.这种以刺激响应型多肽为基础构建的药物诊疗体系,能够在到达肿瘤以前保持药物的零释放,而在靶向到达肿瘤组织后,在肿瘤组织特殊微环境或是外源刺激下(如肿瘤特异性表达酶的刺激、p H刺激和氧化还原刺激等),实现药物的精准靶向释放同时释放出各种诊疗信号.这种具有特异性刺激响应型的多肽载体可以最大程度的提高药物的抗肿瘤效果,降低药物的毒副作用,以及提高肿瘤诊断的精准度.本文简要综述了近年来不同刺激响应型多肽在肿瘤诊疗领域的研究进展.     

联合UV及SDS-PAGE监测并优化碳二亚胺偶联法制备多肽免疫原

建立了一种多肽免疫抗原偶联的双监测法,获得一种操作简便、成功率高、适用范围广的碳二亚胺偶联法,并应用于多种肿瘤相关多肽标志物的免疫抗原制备。以合成多肽SP0104为标准品,牛血清白蛋白(BSA)为载体蛋白,碳二亚胺(EDC)为偶联剂,对加样顺序、投料比、偶联时间等关键条件进行考察,采用UV和SDS-PAGE对反应产物进行联合监测,以偶联比和偶联率联合评价反应条件。加样顺序为多肽和载体蛋白先混合再加到碳二亚胺中,三者质量比为1∶1∶1,偶联时间为12 h,偶联比在4∶1～12∶1之间,偶联率在9%～20%之间,所得SP0104多抗效价达到1∶80 000,MALDI-TOF质谱分析证实该抗体准确捕获目标抗原。用优化的偶联方法制备多肽SPC09、SPC15、SPG01、SPG03、SPG04的免疫抗原,所得免疫血清效价均在1∶1万以上,最高可达1∶51.2万。所建碳二亚胺多肽偶联联合监测法简便实用,可提高多肽抗体制备的成功率。     

基于磁性微球的固定化金属亲和载体及其在蛋白质/多肽纯化中的应用

采用模板法制备的单分散磁性硅胶微球,经过表面修饰偶联上亚氨基二乙酸(IDA),与过渡金属离子Cu2 螯合,制成一种新型的磁性固定化金属亲和纯化载体。用牛血清白蛋白(BSA)作为模型进行磁性固定化金属亲和吸附蛋白的研究,结果表明,BSA在磁性亲和载体上的吸附可用Langmuir吸附方程描述,对BSA的饱和吸附量为90mg/g。将磁性亲和载体用于带有组氨酸标签的镇痛抗肿瘤多肽(analgesic-antitumorpeptide,AGAP)纯化,在未经过滤的细胞裂解液中可以将AGAP一步纯化,非特异性吸附低,操作简便,完全适用于含有组氨酸标记的重组多肽或蛋白的分离纯化。     

DTPA—Eu^3＋螯合物在核酸杂交分析中的应用

报道了靶ＤＮＡ／生物素化λＤＮＡ探针／亲合素／Ｂｉｏ－ＢＳＡ（ＴＧ－Ｅｕ＾３＋体在核酸杂交分析中的应用，亲合素做为连结杂交体与ｉｏ－ＢＳＡ－ＤＴＰＡ－Ｅｕ＾３＋的桥，生物素和ＤＴＰＡ－Ｅｕ＾３＋均标记在载体蛋白（ＢＳＡ，ＴＧ）上，优化了分析条件，比较了己二胺，乙二胺转胺化探针及载体蛋白对杂交分析的影响，方法灵敏度高，重现性较好（ＲＳＤ－５．７％，ｎ＝６）。     

简便高效的超分子多肽治疗策略

在众多疾病治疗药物中,多肽药物以其高活性、特异性与低毒副作用等优点受到广泛关注.然而,稳定性差和体内半衰期短制约了多肽药物的临床发展.最近,国家纳米科学中心易宇、赵宇亮与清华大学王华、张希等合作,提出了一种简便高效的超分子多肽治疗策略.以含有葫芦[7]脲的聚乙二醇为载体,通过N端含苯丙氨酸的多肽与葫芦[7]脲的主客体相互作用,构筑了超分子多肽纳米药物,用于抗肿瘤多肽的体内输送.与单纯多肽药物相比,超分子多肽纳米药物毒副作用减小,在血液循环中稳定性增强,并且更易于在肿瘤部位富集,实现了良好的抑制肿瘤生长效果.该研究为超分子化学在多肽和蛋白类药物递送方面的应用提供了新思路,具有显著的临床转化应用前景.     

DTPA－Eu~（3＋）螯合物在核酸杂交分析中的应用

报道了靶ＤＮＡ／生物素化λＤＮＡ探针／亲合素／Ｂｉｏ－ＢＳＡ（ＴＧ）－ＤＴＰＡ－Ｅｕ~（３＋）体系在核酸杂交分析中的应用。亲合素做为连结杂交体与Ｂｉｏ－ＢＳＡ－ＤＴＰＡ－Ｅｕ~（３＋）或Ｂｉｏ－ＴＧ－ＤＴＰＡ－Ｅｕ~（３＋）的桥，生物素和ＤＴＰＡ－Ｅｕ~（３＋）均标记在载体蛋白（ＢＳＡ，ＴＧ）上。优化了分析条件，比较了己二胺、乙二胺转胺化探针及载体蛋白对杂交分析的影响。方法灵敏度高（１ｐｇ／ｗｅｌｌ靶λＤＮＡ），重现性较好（ＲＳＤ＝５．７％，ｎ＝６）。     

The baculovirus expression system as a tool for generating diversity by viral surface display

It has become a major goal of molecular biologists, biochemists, and immunologists to be able to modulate the structure of proteins, in order to increase their antigenicity, alter their biological properties and/or explore their function. Based on the concept of bacterial phage display, by which proteins are being selected and analyzed in conjunction with their genetic information, eukaryotic systems have been investigated for their use in generating biomolecular diversity. The advantage of posttranslational modification and the possible harbouring of structural complex proteins has lead scientists to include eukaryotic systems in the wide field of molecular design. The ideal expression vectors for surface display are eukaryotic viruses, that allow large gene insertions, efficiently present foreign proteins on the particle surface, are easy to propagate and, if possible, not pathogenic to humans. By inserting peptides into a native virus coat protein or by expressing foreign proteins as coat protein fusion proteins or linked to specific anchor domains it becomes possible to display polypeptides of interest on the surface of replicating particles. A variety of different strategies are currently under investigation in order to utilize the baculovirus insect cell expression system for efficient display on the surface of virus particles as well as on the surface of virally infected insect cells. Increasing the transfection efficiency, optimizing cloning procedures, and establishing applicable selection methods have lead to the development of a powerful tool for drug screening and ligand screening.     

Methods of virus formation

Viruses occur in a great variety of shapes and sizes, but for all their diversity in appearance they possess certain characteristics in common: all viruses contain a single nucleic acid molecule – deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) – surrounded by a protective protein coat. Among other things, the protein coat enables the genetic information stored in the nucleic acid to enter a host cell in a usable state, where it can initiate the reproduction of identical virus particles. After penetration of the cell the foreign genetic material of the virus particle first induces the synthesis of macromolecules not normally present in the cell: the viral nucleic acid undergoes replication and very many copies are produced, the protein of the virus coat is synthesized, and then these components are assembled to form a new generation of infectious virus particles. Most viruses also exhibit certain common structural features: their protein coat is built up from subunits arranged in helical or icosahedral fashion around the genetic material.     

NMR of fd coat protein.

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the 13C and 1H nuclei of the coat protein. Both the 13C and 1H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDA solubilized virus. Natural abundance 13C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The alpha carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the alpha carbons of globular proteins. The 1H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a 1H spectra gives the pKas for the tyrosines.     

Exploring the local conformational space of a membrane protein by site-directed spin labeling

Molecular modeling based on a hybrid evolutionary optimization and an information condensation algorithm, called GHOST, of spin label ESR spectra was applied to study the structure and dynamics of membrane proteins. The new method is capable of providing detailed molecular information about the conformational space of the spin-labeled segment of the protein in a membrane system. The method is applied to spin-labeled bacteriophage M13 major coat protein, which is used as a model membrane protein. Single cysteine mutants of the coat protein were labeled with nitroxide spin labels and incorporated in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers. The new computational method allows us to monitor distributions of local spatial constraints and molecular mobility, in addition to information about the location of the protein in a membrane. Furthermore, the results suggest that different local conformations may coexist in the membrane protein. The knowledge of different local conformations may help us to better understand the function-structure relationship of membrane proteins.     

Filamentous phage studied by magic-angle spinning NMR: resonance assignment and secondary structure of the coat protein in Pf1

Assignments are presented for resonances in the magic-angle spinning solid-state NMR spectra of the major coat protein subunit of the filamentous bacteriophage Pf1. NMR spectra were collected on uniformly 13C and 15N isotopically enriched, polyethylene glycol precipitated samples of fully infectious and hydrated phage. Site-specific assignments were achieved for 231 of the 251 labeled atoms (92%) of the 46-residue-long coat protein, including 136 of the 138 backbone atoms, by means of two- and three-dimensional 15N and 13C correlation experiments. A single chemical shift was observed for the vast majority of atoms, suggesting a single conformation for the 7300 subunits in the 36 MDa virion in its high-temperature form. On the other hand, multiple chemical shifts were observed for the Calpha, Cbeta, and Cgamma atoms of T5 in the helix terminus and the Calpha and Cbeta atoms of M42 in the DNA interaction domain. The chemical shifts of the backbone atoms indicate that the coat protein conformation involves a 40-residue continuous alpha-helix extending from residue 6 to the C-terminus.     

Controlled encapsidation of gold nanoparticles by a viral protein shell

Icosahedral virus capsids demonstrate a high degree of selectivity in packaging cognate nucleic acid components during assembly. This packaging specificity, when integrated as part of a nanotechnological protocol, has the potential to encapsidate a wide array of foreign materials for delivery of therapeutics or biosensors into target cells. Red clover necrotic mosaic virus (RCNMV) exclusively packages two genomic ssRNAs initiated by a specific protein:RNA interaction between the RCNMV coat protein (CP) and the viral RNA origin of assembly (OAS) element. In the present work, an oligonucleotide mimic of the RCNMV OAS sequences is attached to Au nanoparticles as a recognition signal to initiate the virion-like assembly by RCNMV CP. Covalent linkage of the OAS to Au functions as a trigger for specific encapsidation and demonstrates that foreign cargo can be packaged into RCNMV virions.     

利用pⅧ展示系统改进噬菌体抗体芯片

将展示单链抗体的重组噬菌体与羧基终止的硅片偶联, 制成噬菌体抗体芯片, 可用于检测多类蛋白质和蛋白质组. 通常抗体被展示于噬菌体外壳蛋白pⅢ上, 由此制备的芯片灵敏度和信噪比较低. 我们选用凝血酶特异的单链抗体为代表, 比较了pⅢ展示系统和pⅧ展示系统制成芯片的检测效果. 由于pⅧ展示系统的融合蛋白拷贝数多, 所受空间位阻小, 大幅度提高了噬菌体抗体芯片的灵敏度和信噪比, 有望用于制备新型蛋白质芯片.     

Development of a Platform for Noncovalent Coupling of Full Antigens to Tobacco Etch Virus-Like Particles by Means of Coiled-Coil Oligomerization Motifs

Virus-like particles are excellent inducers of the adaptive immune response of humans and are presently being used as scaffolds for the presentation of foreign peptides and antigens derived from infectious microorganisms for subunit vaccine development. The most common approaches for peptide and antigen presentation are translational fusions and chemical coupling, but some alternatives that seek to simplify the coupling process have been reported recently. In this work, an alternative platform for coupling full antigens to virus-like particles is presented. Heterodimerization motifs inserted in both Tobacco etch virus coat protein and green fluorescent protein directed the coupling process by simple mixing, and the obtained complexes were easily taken up by a macrophage cell line.     

Liquid crystal properties of a self-assembling viral coat protein

Monomers of the fd bacteriophage major coat protein, g8p, interact to form a helical bent-core structure surrounding the viral deoxyribonucleic acid (DNA). It was hypothesised that g8p has mesophasic characteristics guiding viral coat assembly. Atomic force and scanning electron microscopy reveal that g8p monomers organise to form films that wrap around a central axis to form birefringent fibres. These fibres resemble the filament structure of bent-core thermotropic B7 liquid crystals. These results offer insight into the g8p self-assembly mechanism that results in the formation of new viral particles in vivo.